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Bombyx mori whole genome knockout vector library based on CRISPR/Cas9 system and construction method

A construction method and a knockout carrier technology, applied in the field of eukaryotic gene knockout

Pending Publication Date: 2020-08-18
SOUTHWEST UNIVERSITY
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Problems solved by technology

[0006] There is no high-throughput research on functional genome technology in silkworm. It is very necessary to construct a genome-wide knockout vector library based on piggyBac system and CRISPR / Cas9 system in silkworm.

Method used

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  • Bombyx mori whole genome knockout vector library based on CRISPR/Cas9 system and construction method
  • Bombyx mori whole genome knockout vector library based on CRISPR/Cas9 system and construction method
  • Bombyx mori whole genome knockout vector library based on CRISPR/Cas9 system and construction method

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Embodiment

[0044] The Bombyx mori embryonic cell line (BmE) used in this example is a commonly used cell line in biological experiments (PMID: 17570024).

[0045] 1. The purpose of the present invention is to provide a vector library for silkworm genome-wide knockout based on the CRISPR / Cas9 system. The process of building a library is as follows: figure 2 shown.

[0046] 2. In order to realize the delivery of the silkworm CRISPR / Cas9 system, the present invention provides a delivery vector pB-CRISPR based on the piggyBac transposon system, its nucleotide sequence is shown in SEQ ID NO.1, and the vector map is shown in figure 1 . details as follows:

[0047] Using the piggyBac transposon system basic vector piggyBacModify (synthesized by Kingsray) as the initial vector, a piggyBac transposon system-mediated CRISPR / Cas9 gene knockout vector backbone was constructed, mainly including piggyBac transposable arms (including two piggyBac Transposon terminal inverted repeat sequence (invert...

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Abstract

The invention relates to a bombyx mori whole genome knockout vector library based on a CRISPR / Cas9 system and a construction method. The vector library comprises a delivery vector system piggyBac transposon vector system used for delivering a CRISPR / Cas9 system and a vector library pB-CRISPR-library composed of sgRNA sequences of targeting sites of genes of all encoding proteins of bombyx mori. According to the invention, based on the strong bearing capacity of the piggyBac transposon system, a Cas9 protein expression cassette and an sgRNA expression cassette, which are two constituent elements of a CRISPR / Cas9 system, can be integrated into one vector; and meanwhile, the vector further comprises a screening marker Zeocin resistance gene expression cassette, and the two parts jointly forman all-in-one vector pB-CRISPR for CRISPR / Cas9 knockout of bombyx mori.

Description

technical field [0001] The invention belongs to the technical field of eukaryotic gene knockout, and relates to a silkworm genome-wide knockout vector library and a construction method based on the CRISPR / Cas9 system. Background technique [0002] Silkworm is not only an important economic insect, but also a model organism of Lepidoptera. The silkworm is one of the earliest organisms to complete the whole genome sequencing. After the completion of the silkworm genome project, the research on the functional genome of the silkworm has become an important direction in the field of silkworm research. At present, genetic manipulation technologies such as transgenic, RNAi, and gene editing have been established for the study of silkworms. Functional genes, however, in the face of the functions of tens of thousands of silkworm protein-coding genes, traditional forward genetics is time-consuming and labor-intensive, and a high-throughput research method is urgently needed to study t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/90C40B50/06C40B40/04
CPCC12N15/85C12N15/902C40B50/06C40B40/04
Inventor 马三垣常珈菘夏庆友
Owner SOUTHWEST UNIVERSITY
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